The overall objective of this MCSDA proposal is to gain research training to develop the applicant into an independent investigator in the area of molecular physiology of the gut. While the applicant has already gained competence in biochemistry, molecular biology and stem cell technology for gene-targeted disruption, he now proposes to apply these tools to study neurotransmitters in the gut and their role in pathophysiology. This entails learning physiological techniques coupled with formal didactic training. In the past, studies in this field have relied on available pharmacologic agents which are often non-specific, non-uniform and transient in their actions. This proposal uses mice with gene-targeted disruption of nitric oxide synthases (NOS) and vasoactive intestinal peptide (VIP) to understand their inhibitory role in gut neurotransmission and motility. Inhibitory junction potentials, mechanical relaxations, immunocytochemical localization, and changes in synthesis or release of putative neurotransmitters will be examined. The specific aims are: (1) to define the role of NO from neuronal constitutive NOS (ncNOS) in inhibitory neurotransmission in the stomach: (2) To define overlapping or compensatory neurotransmitter mechanisms which are present in ncNOS-deficient mice; (3) to define the direct inhibitory effect of VIP on gastric smooth muscle from the ncNOS- and endothelial constitutive NOS- (ecNOS-) deficient mice; and (4) to produce VIP-deficient mice by gene-targeting and to investigate changes in gastric motility and inhibitory neurotransmission due to this disruption. These studies of molecular physiology would elucidate our understanding of neuromuscular transmission not only in the stomach but throughout the gut, and contribute to understanding of neuromuscular pathogenesis and possible treatment of clinical disorders such as achalasia, gastric stasis, congenital pyloric stenosis, intestinal pseudo-obstruction, Hirschsprung's disease, and functional bowel disorders. This award will allow the applicant to develop into a productive independent investigator in the area of molecular physiology involving a multi-disciplinary approach to understanding and treatment of human diseases.